Sigma-E is required for the production of the antibiotic actinomycin in Streptomyces antibioticus

The phsA gene encodes phenoxazinone synthase (PHS), which catalyses the penultimate step in the pathway for actinomycin biosynthesis in Streptomyces antibioticus . The phsA promoter strikingly resembles a putative Streptomyces σ^E cognate promoter, and purified Eσ^E holoenzyme transcribed the phsA promoter in vitro . However, the phsA promoter was still active in an S. antibioticus sigE null mutant and the level of PHS activity was unaffected. Despite this, disruption of sigE blocked actinomycin production completely. The loss of actinomycin production correlated with a 10-fold decrease in the activity of actinomycin synthetase I, the enzyme which catalyses the activation of the precursor of the actinomycin chromophore.     

Integrative, xylE-based promoter probe vectors for use in Streptomyces

Two promoter probe plasmid vectors, designated pIPP1 and pIPP2, were constructed from the existing plasmids pXE4 and pSET152. pIPP1 and 2 use the xylE gene of Pseudomonas putida as a reporter and can be transferred to streptomycetes by conjugation from Escherichia coli. The function of these plasmids as promoter probes was demonstrated in Streptomyces antibioticus and Streptomyces coelicolor using the phenoxazinone synthase and polynucleotide phosphorylase promoters from S. antibioticus. xylE activity could be detected in colonies on agar plates or via the in vitro assay for catechol dioxygenase. The integration into the S. antibioticus chromosome of the constructs containing the phsA promoter was verified by Southern blotting. The presence of the bla locus in pIPP1 allows the recovery of putative promoters by marker rescue.     

Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans

In two separate studies a BclI-generated DNA fragment coding for the enzyme tyrosinase, responsible for melanin synthesis, was cloned from Streptomyces antibioticus DNA into two SLP1.2-based plasmid vectors (pIJ37 and pIJ41) to generate the hybrid plasmids, designated pIJ700 and pIJ701, using S. lividans 66 as the host. The fragment (1.55 kb) was subcloned into the multicopy plasmid pIJ350 (which carries thiostrepton resistance and has two non-essential BclI sites) to generate four new plasmids (pIJ702-pIJ705) with the tyrosinase insert located in either orientation at each site. All six plasmids conferred melanin production (the Mel+ phenotype) on their host. As in the S. antibioticus parent, strains of S. lividans carrying the gene specifying tyrosinase synthesis possessed an enzyme activity which was inducible. Most of the tyrosinase activity was secreted during growth of S. antibioticus; in contrast, the majority remained intracellular in the S. lividans clones. The specific activity of the induced tyrosinase activity (intracellular) was higher (up to 36-fold) when the gene was present on the multicopy vector in comparison with its location on the low copy plasmids, pIJ700 or pIJ701, or in S. antibioticus. Restriction mapping of the tyrosinase fragment in pIJ702 revealed endonuclease cleavage sites for several enzymes, including single sites for BglII, SphI and SstI that are absent from the parent vector (pIJ350). Insertion of DNA fragments at any one of these sites abolished the Mel+ phenotype. The results indicate that pIJ702 is a useful cloning vector with insertional inactivation of the Mel+ character as the basis of clone recognition.     

Actinomycin synthesis in Streptomyces antibioticus. Purification and properties of a 3-hydroxyanthranilate 4-methyltransferase

A methyltransferase, which utilizes 3-hydroxyanthranilic acid (HAA) as a substrate, has been purified to near homogeneity from 30-36-h mycelium of the bacterium Streptomyces antibioticus. The enzyme was obtained in approximately 20% yield with a purification of 130-fold. Polyacrylamide gel electrophoresis under denaturing conditions indicates that the enzyme is composed of a single subunit with Mr of about 36,000. On chromatography in 0.5 M NaCl, the enzyme displays a molecular weight of about 37,000. The specific activity of the enzyme in S. antibioticus mycelium is maximal between 30 and 36 h following inoculation of galactose/glutamic acid medium and, at those times post-inoculation, the specific activity is essentially the same in extracts of mycelium obtained from cultures grown on glucose rather than galactose as the carbon source. The enzyme activity is stimulated by Na2EDTA (in crude extracts) and by 2-mercaptoethanol and the methyltransferase shows a strong preference for HAA as substrate as compared with a number of HAA analogs. Thin layer chromatography of ethyl acetate extracts of large-scale incubation mixtures confirms that the product of the reaction is 4-methyl-3-hydroxyanthranilic acid. The reaction product was also a substrate for phenoxazinone synthase and was incorporated into actinomycin by S. antibioticus mycelium. Kinetic parameters for the methyltransferase reaction was determined.     

Cloning of a DNA fragment from Streptomyces griseus which directs streptomycin phosphotransferase activity

DNA from Streptomyces griseus ATCC 12475 was partially digested with Sau3A and fragments were ligated into BglII-cleaved pIJ702. When the ligation mixture was used to transform protoplasts of Streptomyces lividans TK54, two transformants resistant to both thiostrepton and streptomycin were isolated. The hybrid plasmids pBV3 and pBV4 which they contained, carrying inserts of sizes 4.45 and 11.55 kbp respectively, each retransformed S. lividans to streptomycin resistance at high efficiency. Both plasmids hybridized to restriction digests of S. griseus chromosomal DNA in Southern blot experiments. In vitro deletion and sub-cloning experiments showed the sequence conferring streptomycin resistance to lie within a segment of 1.95 kbp. Extracts of TK54(pBV3) and TK54(pBV4) contained a streptomycin phosphotransferase similar to that in extracts of S. griseus. Streptomycin phosphotransferase activity appeared in extracts of S. griseus, TK54(pBV3) and TK54(pBV4) within 2 d of inoculation. When pBV3 and pBV4 were retransformed into S. griseus with selection for thiostrepton resistance, plasmid DNA of sizes corresponding to the incoming plasmids was found in the transformants. In these transformants the phosphotransferase appeared at 1.5 rather than 2 d, and reached a level over twice that of the original S. griseus strain.     

Novobiocin-resistance sequences from the novobiocin-producing strain Streptomyces niveus

Two distinct DNA sequences expressing novobiocin resistance in Streptomyces lividans were cloned from the novobiocin-producing species Streptomyces niveus. Clone pGL101 (5kb) conferred resistance to 50 micrograms ml-1 novobiocin, whereas clones pGL102 and pGL103, which carry the same 6.5kb insert but in opposite orientations, expressed resistance to 150 micrograms ml-1. The cloned inserts from pGL101 and pGL103 failed to hybridize with each other or with the cloned novobiocin-resistant gyrB sequence from Streptomyces sphaeroides. Both probes hybridized strongly with DNA from the novobiocin-producing species S. niveus and S. sphaeroides but no hybridization (pGL103) or very weak hybridization (pGL101) was detected with DNA from the non-producing species S. lividans, Streptomyces griseus and Streptomyces antibioticus. S. niveus contains at least three novobiocin-resistance determinants with the pGL101 and pGL103 cloned sequences specific for novobiocin-producing strains of Streptomyces.     

Twin-Arginine Translocation Pathway in Streptomyces lividans

The recently discovered bacterial twin-arginine translocation (Tat) pathway was investigated in Streptomyces lividans, a gram-positive organism with a high secretion capacity. The presence of one tatC and two hcf106 homologs in the S. lividans genome together with the several precursor proteins with a twin-arginine motif in their signal peptide suggested the presence of the twin-arginine translocation pathway in the S. lividans secretome. To demonstrate its functionality, a tatC deletion mutant was constructed. This mutation impaired the translocation of the Streptomyces antibioticus tyrosinase, a protein that forms a complex with its transactivator protein before export. Also the chimeric construct pre-TorA-23K, known to be exclusively secreted via the Tat pathway in Escherichia coli, could be translocated in wild-type S. lividans but not in the tatC mutant. In contrast, the secretion of the Sec-dependent S. lividans subtilisin inhibitor was not affected. This study therefore demonstrates that also in general in Streptomyces spp. the Tat pathway is functional. Moreover, this Tat pathway can translocate folded proteins, and the E. coli TorA signal peptide can direct Tat-dependent transport in S. lividans.     

Structure of phenoxazinone synthase from Streptomyces antibioticus reveals a new type 2 copper center

The multicopper oxidase phenoxazinone synthase (PHS) catalyzes the penultimate step in the biosynthesis of the antibiotic actinomycin D by Streptomyces antibioticus. PHS exists in two oligomeric forms: a dimeric form and a hexameric form, with older actinomycin-producing cultures containing predominately the hexameric form. The structure of hexameric PHS has been determined using X-ray diffraction to a resolution limit of 2.30 A and is found to contain several unexpected and distinctive features. The structure forms a hexameric ring that is centered on a pseudo 6-fold axis and has an outer diameter of 185 A with a large central cavity that has a diameter of 50 A. This hexameric structure is stabilized by a long loop connecting two domains; bound to this long loop is a fifth copper atom that is present as a type 2 copper. This copper atom is not present in any other multicopper oxidase, and its presence appears to stabilize the hexameric structure.     

relA Is Required for Actinomycin Production in Streptomyces antibioticus

The relA gene from Streptomyces antibioticus has been cloned and sequenced. The gene encodes a protein with an Mr of 93,653, which is 91% identical to the corresponding protein from Streptomyces coelicolor. Disruption of S. antibioticus relA produces a strain which grows significantly more slowly on actinomycin production medium than the wild type or a disruptant to which the intact relA gene was restored. Moreover, the disruptant was unable to accumulate ppGpp to the levels observed during the normal course of growth and actinomycin production in the wild type. The strain containing the disrupted relA gene did not produce actinomycin and contained significantly lower levels of the enzyme phenoxazinone synthase than the wild-type strain. Actinomycin synthetase I, a key enzyme in the actinomycin biosynthetic pathway, was undetectable in the relA disruptant. Growth of the disruptant on low-phosphate medium did not restore actinomycin production.     

Structural instability of a bifunctional plasmid pZG1 and single-stranded DNA formation in Streptomyces

Structural instability of a hybrid plasmid pZG1, consisting of Escherichia coli pBR322 and Streptomyces pIJ350 plasmids, has been studied in Streptomyces. After transformation of S. lividans 1326 and S. rimsus R6 protoplasts with pZG1, transformants harbored the intact pZG1 and various deleted plasmid forms. The pattern of deleted plasmids varied with the transformant colony age and changed upon subcultivation. The presence of intact pZG1 in at least a part of the Streptomyces colonies indicated that the plasmid was capable of replicating in the transformants and that deletion events occurred after at least one round of replication. Less instability of pZG1 in S. rimosus R6 was observed when this strain was transformed with the DNA isolated from the same strain. pZG1 and its various derivatives were found in S. lividans 1326 and S. rimosus R6 as double- and single-stranded DNA molecules. Structural instability of pZG1 could therefore be due at least in part to the presence of single-stranded DNA.     

Cloning and expression of Mycobacterium bovis BCG DNA in "Streptomyces lividans"

The ability of "Streptomyces lividans" to use the expression signals of genes from Mycobacterium bovis BCG was tested in vivo by using gene fusions. Random DNA fragments from M. bovis BCG were inserted into promoter-probe plasmids in Escherichia coli and in "S. lividans." Comparison with promoter activity detected with random DNA fragments from the respective hosts suggested that "S. lividans" efficiently utilizes a high proportion of mycobacterial promoters, whereas a smaller fraction are expressed, and expressed more weakly, in E. coli. M. bovis BCG DNA fragments were also inserted into the specially constructed translational fusion vector (pIJ688) in "S. lividans." pIJ688 contains the kanamycin phosphotransferase gene (neo) from transposon Tn5, truncated at its amino terminus, as the indicator. The results suggested that "S. lividans" uses M. bovis BCG translational signals almost as efficiently as its own signals. Moreover, several hybrid proteins with an M. bovis BCG-derived amino terminus seemed to be reasonably stable in "S. lividans." These experiments indicate that "S. lividans" may be a suitable host for the expression of Mycobacterium leprae and Mycobacterium tuberculosis genes from their own signals. This is a precondition for the expression of entire biosynthetic pathways, which could be valuable in the production of diagnostic and therapeutic agents. The vectors may also have wider applications for the analysis of gene expression in Streptomyces.     

Development of integrative vectors for Actinomycetes--producers of antibiotics]

The integrative vectors pSU 475 and pSU 476 with variable numbers of copies per genome were developed for antibiotic producing actinomycetes. For this, the amplifying sequence AUD-Sr 1 of Streptomyces rimosus and the BamHIB fragment of the eSA 1 genetic element from Streptomyces antibioticus were used. The eSA 1 fragment was an element required for integration of a vector to the actinomycete chromosomes since it was homologous with the chromosomal DNAs of S. lividans, S. erythraeus and S. antibioticus. At the first stage the AUD-Sr 1 sequence within the actinomycete plastid pSU 23 was cloned by the vector pUC 19 to E coli. In that experiment the 12.4-kb plasmid pSU 449 was isolated. At the second stage the BamHIB-fragment of the eSA 1 element was incorporated into the resultant hybrid plasmid pSU 449. The 16.5-kb hybrid plasmids pSU 475 and pSU 476 were isolated. In these plasmids the BamHIB fragment of eSA 1 was present in two orientations. The developed vectors were useful in cloning DNA to S. lividans and S. erythraeus.     

Site-specific degradation of Streptomyces lividans DNA during electrophoresis in buffers contaminated with ferrous iron.

Streptomyces lividans DNA contains a modification which makes it susceptible to double-strand cleavage during electrophoresis in buffers contaminated with ferrous iron (which may be present in some batches of EDTA). The cleavage of the DNA is site-specific and the average fragment size resulting from limit digestion of total S. lividans DNA is about 6kb. DNA from Streptomyces coelicolor A3(2) and several other Streptomyces strains, and from E. coli, is not cleaved under the same conditions. A S. lividans mutant has been isolated which lacks the DNA modification. We suspect that many reports of "poor" preparations of S. lividans plasmids may be due to the above effect.     

Streptomyces ATP nucleotide 3'-pyrophosphokinase and its gene.

Streptomyces ATP nucleotide 3'-pyrophosphokinase is an extracellular, ribosome-independent, and stringent factor-mimic ppGpp synthetase with an unusually broad acceptor spectrum. The gene-containing DNA fragments cloned from chromosomal DNA of a producer S. morookaensis into pIJ699 and pUC plasmids were found to express the active enzyme in the transformed S. lividans TK24 and enteric E. coli JM109 and nitrogen-fixing Klebsiella pneumoniae M5a1 and 5022, respectively. Base sequence of the structural gene and the deduced amino acid sequence exhibited little homology to those of E. coli stringent factor and related proteins. Growth retardation was seen in some transformants.